I have tried using 12% resolving gel as well as 10% resolving gel and 5% stacking gel combination. I have also run the marker which separated fine. But why can I not see bands for my sample? i have used 1mg/ml concentration of my hydrolysate with 2X laemmeli buffer in the ratio 1:1. Also I have added mercaptoethanol and heated the sample at 95Deg C for 5 mins. What maybe the issue can anybody suggest?
Since your ladder shows proper separation, the issue likely lies with your sample. Does the protein of interest separate properly prior to hydrolysis? For your current samples, consider increasing the sample load to 30-50 µg total protein to see if any bands become visible on the total protein stain.
It also appears that you’re cutting off the loading wells. Next time, try transferring with the wells intact; if the protein is running poorly, it may be visible just below the loading wells in the stacking gel. By the way, what is the size of the protein you’re working with?
Anton Lennikov Thank you for your attention and your suggetion. I haven't really fractionated my sample so am unsure about the size of my hydrolysates. Am running both hydrolysed and unhydrolysed sample at the same time. But as per literature am expecting the range between 10-250 kD. i was staining my sample for 2 hrs only and before that I fixed it for an hour. Do you think the exposed staining time might be an issue? or maybe the voltage and current applied?
Darshana Deka
If your hydrolysed protein is not separating on SDS-PAGE, consider the following adjustments:
Peptide Size: Hydrolysis may have produced tiny peptides. For increased resolution, use a 15% gel or Tricine-SDS-PAGE.
Sample Dilution: 1 mg/mL could be overly concentrated. Dilute to 0.2-0.5 mg/mL to prevent spreading.
Buffer and Heating: Double-check your Laemmli buffer preparation and heat samples to 95°C for optimal denaturation.
Run Settings: Adjust the voltage and run time as needed.
These modifications should help improve separation.
Hameer Khan Khaskheli Hi, thank you for your suggestions. i have tried using 15% gel also tried with 0.5mg/ml concentration as well as 5mg/ml concentration. But didn't work. Even though if the sample was overly concentrated, at least I should see smears if not defined bands. But I could observe nothing.
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